Validation of Lucigenin as a Chemiluminescent Probe to Monitor Vascular Superoxide as Well as Basal Vascular Nitric Oxide Production

• Published in: Biochemical and biophysical research communications Vol. 254; no. 2; pp. 319 - 324
• Main Authors: Skatchkov, Mikhail P., Sperling, Daniel, Hink, Ulrich, Mülsch, Alexander, Harrison, David G., Sindermann, Irene, Meinertz, Thomas, Münzel, Thomas
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 19.01.1999
• Subjects: Acetylcholine - pharmacology; Acridines - pharmacology; Animals; Aorta - drug effects; Aorta - physiology; Aorta - physiopathology; Electron Spin Resonance Spectroscopy - methods; Endothelium, Vascular - physiology; Endothelium, Vascular - physiopathology; Hypercholesterolemia - physiopathology; In Vitro Techniques; Luminescent Measurements; Muscle, Smooth, Vascular - drug effects; Muscle, Smooth, Vascular - physiology; Muscle, Smooth, Vascular - physiopathology; Nitric Oxide - analysis; Nitric Oxide - metabolism; omega-N-Methylarginine - pharmacology; Pentetic Acid - pharmacology; Rabbits; Reproducibility of Results; Scintillation Counting - methods; Superoxide Dismutase; Superoxides - analysis; Superoxides - metabolism; Vasodilation - drug effects; Vasodilation - physiology
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.1998.9942

Lucigenin has been widely used as a chemiluminescent substrate to monitor vascular superoxide (O•−2) formation. The validity of lucigenin for detection of O•−2has been questioned because O•−2is generated by lucigenin itself. It has been shown that the concentration of lucigenin is a critical parameter affecting the validity of this assay. In the present studies we evaluated a reduced concentration of lucigenin (5 μM) as a tool to quantify O•−2production in vascular tissue. Lucigenin-induced effects on endothelial function were assessed by isometric tension recording of isolated aortic rings suspended in organ baths. The effects of lucigenin on O•−2production were studied using spin trapping and electron spin resonance spectroscopy. Lucigenin at 250 μM but not at 5 μM caused a significant attenuation of endothelium-dependent relaxations to acetylcholine, which was prevented by pretreatment with superoxide dismutase. Spin-trapping studies revealed that lucigenin at 250 μM increased vascular O•−2production several fold while 5 μM lucigenin did not stimulate O•−2production. Inhibition of NO synthase byNG-momomethyl-l-arginine as well as the removal of the endothelium almost doubled lucigenin-derived chemiluminescence (LDCL), indicating that basal production of endothelium-derived NO depresses the baseline chemiluminescence signal. Thus, lucigenin at a concentration of 5 μM seems to be a sensitive and valid probe for assessing O•−2in vascular tissue. It can also be used as an indirect probe to estimate basal vascular NO release.